The main goal of this proposal is to study the functional consequences and mechanisms underlying antidepressant-induced down-regulation of the serotonin transporter (SERT) and norepinephrine transporter (NET). These proteins are the key cellular targets for drugs used in the treatment of depression. Our general hypothesis is that the time-dependent antidepressant-induced decrease in these transporters may be one of the crucial determinants of both why and when serotonergic and noradrenergic transmission is markedly enhanced, with consequent behavioral improvement. To study this, both time course and mechanistic studies will be carried out. Time course studies will correlate the time-dependent sertraline-induced changes in SERT density (as measured by quantitative autoradiography) with changes in the chronoamperometric signal caused by local application of 5-HT in brain. This will be done after cessation of treatment also. Non-linear regression techniques will be applied to the electrochemical signal to obtain Vmax and KT values for 5-HT clearance by the SERT. The time course of down-regulation of the NET caused not only by DMI but also reboxetine and venlafaxine, at doses producing steady-state serum levels at the low and high ends of its therapeutic range, will be examined, as will the time course of the return of NET binding sites after cessation of treatment. At times when NET binding sites are down-regulated, function of the NET will be assessed by measuring the uptake of 3H-NE in synaptosomes. Different approaches will be used to examine the mechanisms involved in these phenomena. We will study the time course of changes in mRNA for the SERT and its binding sites and mRNA for the NET and its binding sites during treatment of rats with antidepressants and after their cessation. We will study if chronic treatment of rats with uptake inhibitors either alters the distribution or decreases SERT or NET protein in purified membrane fractions obtained from brain. Purified membrane fractions will be obtained by ultracentrifugation of membranes layered on a sucrose gradient and the amount of SERT or NET protein in these fractions measured by Western blot analysis with specific antibodies. Knockout mice will be used to determine influences of PKC epsilon on antidepressant-induced down-regulation of the SERT and NET. Mice lacking 5-HT1B receptors will be used to see if they exhibit altered sertraline-induced down-regulation of the SERT. These experiments involve multiple approaches to study an effect of specific antidepressant drugs that may be very important in sustaining the enhancement of monoaminergic transmission that underlies clinical improvement